WO2025090030A1 - A novel method for preparing a hypoallergenic nutritional product suitable for subjects with cow's milk allergy - Google Patents

Abstract

A novel method for preparing a hypoallergenic nutritional product suitable for subjects with cow's milk allergy The present invention discloses a hypoallergenic nutritional product suitable for subjects with cow's milk allergy, and a novel method for preparation thereof.

Description

A novel method for preparing a hypoallergenic nutritional product suitable for subjects with cow's milk allergy

FIELD OF THE INVENTION

The present invention discloses a hypoallergenic nutritional product suitable for subjects with cow's milk allergy, and a novel method for preparation thereof.

BACKGROUND

Cooking or flavoring processes (heat, fermentation, and matrix effect) may reduce the allergenicity of the proteins; thus, the frequency and the severity of the allergic reactions can be reduced. The recent studies showed that 70-80% of the children with milk allergy are reactive to pasteurized cow's milk but may tolerate conventional baked milk products (matrix with wheat, sugar and baked at 180- 200°C for 30 minutes, examples are muffin, cake) [1].

The effect of heat on allergens is variable and depends on the food. Casein and a- lactalbumin are more heat resistant than B-lactoglobulin and bovine serum albumin. Boiling the milk at 120°C for 15 minutes does not impair the antigenic properties of casein proteins. The casein has linear epitopes and the protein chains that break down into smaller pieces when denatured by heat maintain their epitope property and continue to bind to IgE. However, B-lactoglobulin has conformational epitopes and loses its ability to bind IgE when denatured with heat. Thus, exposure to high (200°C) and prolonged (30 minutes) temperatures, such as baking, leads to the destruction of conformational epitopes and deterioration of the three-dimensional structure of allergens, thereby reducing the allergenicity of the protein [2] . High pressure also causes structural changes such as denaturation of whey proteins and allows enzymes to access potential hydrophobic sites. At the same time, aggregates formed by enzymatic degradation of allergenic regions reduce the allergenic potential of milk proteins [3], Because of the effects that disrupt such antigenic properties, many milk allergic patients can tolerate baked milk products.

Another effect that contributes to baked food tolerance is the matrix effect [4] . It is thought that interactions among protein, fat and sugar in the cake matrix reduce IgE binding by closing allergen epitopes. Moreover, if proteins are exposed to heat in the presence of sugar, a reaction called "Maillard reaction" or "glycation" occurs, which modifies the structure and the aggregation/allergenic effects of milk proteins in different ways depending on the different combinations of protein, sugar and temperature [5; 6] . By doing so, the Maillard reaction gives brown colour, flavour, and aroma to the nutrition. Glycation stands for the breakdown of proteins without enzymes, therefore Maillard reaction also disrupts epitopes by degrading the proteins.

Baked milk products are less allergenic because of the heat and matrix effects as mentioned earlier. There are studies showed that tolerance to baked-milk products in children with IgE-mediated milk allergy is a good predictor of unheated-milk tolerance development [1; 7; 8; 9]. About 75% of children with milk allergies can tolerate high heat-treated baked goods. Patients with milk allergy reactive to baked milk products constitute the severe phenotype group. More crucially, compared to a strict avoidance diet, which is now the "gold standard of care," adding baked-milk products to the diet appears to significantly accelerate the development of tolerance to unheated-milk. Furthermore, introducing dietary baked milk into the diet is both safe and convenient. Patients and their parents are eager and enthusiastic about this approach. The introduction of baked-milk products into the diet of milk-allergic children marks a significant shift in the treatment paradigm for milk allergies. Due to the risk of anaphylaxis in children who are reactive to baked-milk products, such interventions should only be added under the guidance of a food allergy specialist [9], Before mentioned clinical observations gave raise a sequence called "milk ladder" for the reintroduction of milk and dairy products into the diets of infants and children with mild to moderate milk allergy. It is a treatment approach which is defined as the gradual stepwise reintroduction of the milk allergen from the least allergenic forms, progressing to the most allergenic forms of milk into the diet of the patients.

Currently, the most less hypoallergenic food of the milk ladder is conventional cake. This cake by denaturation effect of high temperature (200°C} and matrix effect of sugar and wheat is less allergenic compared to cheese or milk. At present, there is not an option as a less hypoallergenic product than conventional baked milk product (cake, muffin) to induce de sensitization by oral route for the patients who are reactive to baked milk. The new product that we developed, the “ISAKU”, by denaturation of high temperature, matrix effect and additionally Maillard (glycation) effect (120°C for 3 hours) has less allergenic milk proteins than conventional cake.

There are also other technical solutions for producing hypoallergenic food products for subjects allergic to cow's milk, such as using microorganisms (EP2678419B1) and milk protein hydrolysate preparation by enzymatic reactions (W09700078A1).

Accordingly, there is a need in the art for new and improved hypoallergenic food products for patients allergic to cow's milk, including baked cakes, and methods of making them that address these problems, and it is to these objects that the present invention is directed.

SUMMARY OF THE INVENTION

The present invention relates to a novel method for preparing a hypoallergenic nutritional product suitable for patients with cow's milk allergy.

The present invention also relates to a novel method for preparing a less hypoallergenic nutritional product than conventional baked milk product (cake, muffin) to induce desensitization by oral route for the patients/subjects who are reactive to baked milk.

According to a first aspect of the invention there is provided a method for preparing a hypoallergenic nutritional (food) product that contains hypoallergenic milk proteins with low reaction risk.

Another aspect of the present invention is to provide a method for preparing a less hypoallergenic milk product than conventional baked milk product, with the effect of high and longer duration of temperature and different mixtures of matrix.

The other aspect of the present invention is to provide a method for preparing a hypoallergenic milk product having a lower concentration of total protein and allergenic proteins (casein (a-Sl casein, a-S2 casein, B casein and K casein) and B- lactoglobulin) than the total protein and allergenic proteins in conventional cake or cow's milk.

Accordingly, a broad embodiment of the invention is directed to a method for preparing a hypoallergenic nutritional product suitable for subjects with cow's milk allergy, comprising the steps:

- preparation of dough,

- incubation of the said dough,

- the first baking step (the conventional baking step),

- cooling down of the cake prepared at the first baking step,

- slicing the said cake,

- the second baking step (the additional baking step) of the cake slices.

The invention provides a method that has two steps of baking: the first baking step and an additional (the second) baking step. It is found that the baking time in the second baking step plays an important role in the achievement of the technical effect of the invention.

The present invention also provides a hypoallergenic nutritional milk product suitable for subjects with cow's milk allergy that is prepared with the said method. This newly developed hypoallergenic product (named “ISAKU”) can be used for induction of desensitization because it contains milk proteins.

This object and other objects of this invention become apparent from the detailed discussion of the invention that follows.

Brief Description of Figures

The present invention is illustrated in the accompanying figures wherein;

Figure 1 is an illustration of SDS-PAGE and Bradford Assay results. A- Commassie staining of SDS-PAGE for the proteins of the human breast milk and cow's milk (BM1: first breast milk sample, BM2: second breast milk sample, CM1: first cow's milk sample, CM2: second cow's milk sample, M: marker) B- Comparison between the total protein concentrations of different milk products measured by the Bradford Assay (n=5). C- and D- Casein band intensity analysis of milk, cake and ISAKU samples done with Image Lab, Biorad (n=5, 1: Cow's milk, 2: Baked Cow's milk, 3: Cake, 4: ISAKU Ih, 5: ISAKU 2h, 6: ISAKU 3h). *Comparison of cake and ISAKU-3h; comparison of cow's milk and ISAKU-3h by nonparametric post-hoc analysis.

Total protein concentration and casein band intensity in ISAKU was lower than the conventional cake.

Figure 2 is an illustration of detailed examination of protein contents of cake and ISAKU. A- LC-qTOF-MS analysis of the cake and ISAKU samples (n=5, ISAKU samples were normalized to milk. Analysis was done with the Related-Samples Friedman's Two-Way Analysis of Variance by Ranks) B- a-Sl casein concentration was evaluated by commercial ELISA kit for milk, cake and ISAKU 3h (n=3)

Proteomics results showed that the intensities of intact milk proteins decrease as the cooking time increases.

Figure 3 is an illustration of binding intensity of specific IgE to caseins by western blot in ISAKU, conventional cake (baked milk) and pasteurized milk samples. A- PVDF membrane incubated with 3 patients' sera with slgE higher than 100 kU/L, and negative control (n=4), D- Casein band analysis of milk, cake and ISAKU with Image Lab (n=4), *cake and ISAKU, cow's milk and ISAKU- nonparametric post-hoc analysis, CM: Pasteurized cow's milk, M: Marker

Milk slgE of the milk allergic patients bound incompetently to casein bands of ISAKU.

Figure 4 is an illustration of Binding intensity of specific IgE to whole milk proteins by in-house indirect ELISA in ISAKU and conventional cake (baked milk) samples; the cake/lSAKU specific IgE in-house indirect ELISA results of 35 milk allergic patients, * Wilcoxon matched-pairs signed rank test.

Lower slgE binding signals were obtained with ISAKU compared to the cake.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, it is aimed to discover to a novel method for preparing a hypoallergenic nutritional product suitable for subjects with cow's milk allergy using a novel baking approach and suitable for desensitization of cow's milk allergy. As a result, the effects of present invention have been demonstrated experimentally.

As used herein, the terms "conventional cake" or “conventional baked milk product(s)” refers to a cake preparing by a method that is a conventional known cake preparation method with just one step of baking minimum at 180°C degree. The terms "cake" or "ISAKU" or "hypoallergenic nutritional product" or "hypoallergenic milk product" or "hypoallergenic food product" are used interchangeably for the less hypoallergenic milk product produced by the method subject to the invention, which was also referred to as “ISAKU” during the experimental studies.

In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the present invention relates to a method for preparing a hypoallergenic nutritional milk product suitable for subjects with cow's milk allergy, comprising the steps:

- preparation of dough by mixing at least two dry ingredients and at least one liquid,

- incubation of the said dough in the freezer (-20°C) for one night,

- baking the said dough minimum at 180°C for 30 minutes (the first baking step),

- cooling down of the baked cake prepared at the first baking step,

- slicing the said cake,

- baking the said cake slices at 120°C for 3 hours (the second/additional baking step)

In one embodiment, the present invention provides a dough preparation step that comprises mixing the dry ingredients and adding the liquids to prepare the dough. In other embodiment, the dry ingredients of dough preparation step comprise at least two ingredients. In a preferred embodiment, the dry ingredients comprise flour, a leavening agent, and sugar.

Accordingly, flour, a leavening agent, and sugar components are provided together in a dry ingredients mixture, and then dry ingredients mixture is mixed with liquids in dough preparation step.

According to the present invention, in the preparation of dough step, the flour can be selected from the group consisting of white wheat flour. The leavening agent according to the present invention can be selected from the group consisting of baking powder (sodium bicarbonate). Moreover, the selected oil was sunflower seed oid.

In another embodiment, the added liquid to prepare the dough is milk, preferably cow’s milk.

In a preferred embodiment, the prepared dough in the dough preparation step is poured into stretch film and wrapped. Then, the said dough is kept in the freezer (- 20°C) for one night in the incubation step.

According to the present invention, the method of making a hypoallergenic nutritional product suitable for subjects with cow's milk allergy has two steps of baking. The first step is the classical/conventional baking of the overnight frozen cake minimum at 180°C for 30 minutes. And the second step is a novel additional baking step of the cake slices at 120°C for 3 hours. In the present invention, it was discovered that the additional second baking step plays an important role on lowering the concentration of total protein and allergenic proteins of the cow milk with baking time (3h) and temperature (120°C). In a preferred embodiment, after the first baking step the cake prepared is cooled down and then sliced.

According to the present invention, the second baking step is an additional step and this step comprises baking the cake slices at 120°C for 3 hours.

Furthermore, the method for preparing a hypoallergenic nutritional product suitable for subjects with cow's milk allergy, which has two steps of baking, comprises the steps below:

- mixing the dry ingredients and adding at least one liquid to prepare a dough (dough preparation step),

- pouring said dough into stretch film and wrapping,

- keeping it in the freezer (-20°C) for one night (incubation step),

- heating the oven to 180°C with fan (top and bottom open in fan mode),

- After the oven is heated, removing the stretch of the dough and putting it in the oven (with the cake in ice form),

- baking minimum at 180°C for 30 minutes (the first baking step),

- waiting for 1-2 hours for cooling down the cake,

- slicing the cake after cooling down,

- turning on the cooled oven again at 120°C, but with the fan turned off and the upper and lower heat turned on,

- baking the cake slices in the oven at 120°C for 3 more hours (the second baking step)

- turning upside down slices every half hour,

- after 3 hours, taking out the cake and leaving to cool down,

- pounding the slices in a mortar and ground into a powder like breadcrumbs (optional). All of the various embodiments of the present invention as disclosed herein relates to methods of producing a more hypoallergenic milk product than a conventional cake for patients/subjects who are reactive to baked milk and have severe cow's milk allergy.

In certain other non-limiting embodiments, a hypoallergenic nutritional milk product prepared according to the present invention can be selected from the group consisting of a cake, a cookie, a muffin and a brownie. The hypoallergenic nutritional milk product compositions of the present invention can comprise a variety of different product forms, including almost all conventional known food product form.

These examples are intended to representative of specific embodiments of the invention and are not intended as limiting the scope of the invention.

SPECIFIC EMBODIMENTS

In these embodiments, a hypoallergenic nutritional product was prepared that contains hypoallergenic milk proteins with low reaction risk. Then, all in vitro experiments were performed. Bradford assay, ELISA and proteomics were performed to determine the total protein concentration and milk protein content [casein (a-Sl casein, a-S2 casein, B casein and K casein) and B-lactoglobulin] of hypoallergenic product. Western blot and in-house indirect ELISA were performed to detect slgE antibodies against less hypoallergenic baked milk product.

Examples

Example 1 Development of a new hypoallergenic product

To develop a less hypoallergenic milk product than conventional baked milk product with the effect of high and longer duration of temperature and different mixtures of matrix were tested.

Example 2 Determining the total protein concentration and milk protein content

The total protein concentrations were evaluated with Bradford assay. Then the total proteins were run in SDS-PAGE and casein band intensities were compared among all milk products (milk, baked cake, and hypoallergenic product candidate) (Figure 1). Moreover, the milk protein content [casein (a-Sl casein, a-S2 casein, B casein and K casein) and B-lactoglobulin] was checked by proteomics analysis. The concentration of a-Sl casein was checked with commercial ELISA (Figure 2).

Example 3 Evaluation of IgE reactivity

Sera samples of milk allergic subjects were used to check the IgE reactivity against hypoallergenic product via western blot (Figure 3) and in-house ELISA (Figure 4). Results

Total protein concentration and casein band intensity in ISAKU was lower than the conventional cake

Human breast milk (BM) and CM were run in SDS-PAGE and stained to visualize the protein content (Figure 1A). The protein bands in Figure 1A showed that casein content of CM and BM were different and there weren’t any P-Lactoglobulin bands in BM.

Milk protein concentrations from five different milk products (CM, conventional milk cake, ISAKU Ih, 2h, and 3h) were shown in quintuplicate. The concentration of the ISAKU 3h was significantly lower than the conventional milk cake (p=0.002, Figure IB, 25% low). Based on these concentrations, the same amount of protein was used in the following experiments. Noticeably, the casein band intensity decreased as the cooking time increased for biscotti samples (Figure 1C). We observed that casein band intensities of biscotti 3h were significantly lower than the CM and conventional milk cake, respectively (p=0.008, p=0.014 Figure ID, 95% low).

Proteomics results showed that the intensities of intact milk proteins decrease as the cooking time increases

The proteomics analysis by UC-qTOF-MS showed that the intensities of each casein fraction and P-lactoglobulin were decreased as the cooking time increased. The reduction of intensities was found to be statistically significant for each casein fraction and P-lactoglobulin (Figure 2A). The lowest level of intact allergenic protein was always observed in ISAKU 3h. To find the concentration of a-Sl- casein, EUISA was performed and a-Sl-casein concentration level in ISAKU 3h samples was found significantly lower than conventional cake (p=0.0171, Figure 2B). Milk slgE of the milk allergic patients bound incompetently to casein bands of

ISAKU

Serum of the patients who have CM slgE greater than 100 kU/L were used to analyze the binding capacity of CM slgE to milk proteins obtained from the pasteurized CM, conventional cake, and ISAKU 3h (Figure 3A). Milk slgE binding to caseins was significantly lower in ISAKU 3h than conventional cake and pasteurized CM, respectively (n=4, p= 0.0098, p=0.03, Figure 3B).

Lower OD values were obtained with ISAKU 3h compared to the cake

To investigate the reactivity of the milk-allergic patients’ sera with cake and ISAKU 3h, indirect in-house ELISA was conducted. According to the results of checkerboard titrations of the conventional cake and ISAKU 3h samples done with the patients’ sera whose milk slgE levels >100 kU/L shown in Table 1, the dilution of 1 :20 was used for all samples. The binding of slgE from 31 CM-allergic patients was significantly lower in ISAKU 3h than in conventional milk cake (p<0.0001, Figure 4).

Table 1. Checkerboard titration analysis to determine optimal dilutions of patients' sera and anti-IgE secondary antibody for both conventional cake and ISAKU samples (n=3).

REFERENCES

[1]. Sackesen, C., Suarez-Farinas, M., Silva, R., Lin, J., Schmidt, S., Getts, R., .

. . Sampson, H. A. (2019). A new Luminex-based peptide assay to identify reactivity to baked, fermented, and whole milk. Allergy, 74(2), 327-336. doi:10.1111/all.13581

[2]. Thomas, K., Herouet-Guicheney, C., Ladies, G., Bannon, G., Cockbum, A., Crevel, R., . . . Vieths, S. (2007). Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report. Food Chem Toxicol, 45(7), 1116-1122. doi: 10. 1016/j .fct.2006.12.016

[3]. Penas, E., Prestamo, G., Luisa Baeza, M., Martinez-Molero, M. L, &

Gomez, R. (2006). Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins. International Dairy Journal, 76(8), 831-839. doi:https://doi.org/10. 1016/i.idairyj.2005,08.009

[4]. Nowak-Wegrzyn, A., & Fiocchi, A. (2009). Rare, medium, or well done?

The effect of heating and food matrix on food protein allergenicity. Curr Opin Allergy Clin Immunol, 9 3), 234-237. doi: 10.1097/ACI.0b013e32832b88e7

[5]. Pastorello, E. A., Farioli, L., Conti, A., Pravettoni, V., Bonomi, S., lametti,

S., . . . Ortolani, C. (2007). Wheat IgE-mediated food allergy in European patients: alpha-amylase inhibitors, lipid transfer proteins and low-molecular- weight glutenins. Allergenic molecules recognized by double -blind, placebo-controlled food challenge. Int Arch Allergy Immunol, 144(1), 10-22. doi: 10.1159/000102609

[6]. Teodorowicz, M., Van Neerven, J., & Savelkoul, H. (2017). Food Processing: The Influence of the Maillard Reaction on Immunogenicity and Allergenicity of Food Proteins. Nutrients, 9(8), 835. Retrieved from https://wTvw.mdpi.eom/2072-6643/9/8/835 [7]. Kim, J. S., Nowak-Wcgrzyn. A., Sicherer, S. H., Noone, S., Moshier, E. L., & Sampson, H. A. (2011). Dietary baked milk accelerates the resolution of cow's milk allergy in children. J Allergy Clin Immunol, 128(4), 125- 131.el22. doi: 10. 1016/j.jaci.2011.04.036 [8]. Leonard, S. A., & Nowak-Wcgrzyn. A. H. (2016). Baked Milk and Egg

Diets for Milk and Egg Allergy Management. Immunol Allergy Clin North Am, 36(1), 147-159. doi: 10.1016/j.iac.2015.08.013

[9]. Vilar, L. K., Araujo, F. A., Santos, T. P., Menezes, T. T., Cheik, M. F., & Segundo, G. R. S. (2021). Baked Tolerance in Cow's Milk Allergy: Quite Frequent, Hard to Predict! Int Arch Allergy Immunol, 182(4), 319-323. doi: 10.1159/000511148

Claims

1. A method for preparing a hypoallergenic nutritional milk product suitable for subjects with cow's milk allergy, which has two steps of baking, comprising the steps: a. preparation of dough by mixing at least two dry ingredients and at least one liquid, b. incubation of the said dough in the freezer (-20°C) for one night, c. baking the said dough minimum at 180°C for 30 minutes (the first baking step), d. cooling down of the baked cake prepared at the first baking step (c), e. slicing the said cake, f. baking the said cake slices at 120°C for 3 hours (the second baking step)

2. The method for preparing a hypoallergenic nutritional milk product according to claim 1 , wherein dry ingredients comprise flour, a leavening agent, and sugar and liquid comprises oil and cow’s milk.

3. The method for preparing a hypoallergenic nutritional milk product according to claim 2, wherein said flour is selected from the group consisting of white wheat flour.

4. The method for preparing a hypoallergenic nutritional milk product according to claim 3, wherein said leavening agent is selected from the group consisting of baking powder (sodium bicarbonate).

5. The method for preparing a hypoallergenic nutritional milk product according to claim 4, wherein said oil is vegetable sunflower seed oil.

6. The method for preparing a hypoallergenic nutritional milk product according to any of the preceding claims, the said liquid added to the dry ingredient mixture in step (a) is cow’s milk and vegetable sunflower seed oil. .

7. The method for preparing a hypoallergenic nutritional milk product according to any of the preceding claims, comprises the steps below:

- mixing the dry ingredients and adding at least one liquid to prepare a dough,

- pouring said dough into stretch fdm and wrapping,

- keeping it in the freezer (-20°C) for one night,

- heating the oven to 180°C with fan (top and bottom open),

- After the oven is heated, removing the stretch of the dough and putting it in the oven (with the cake in ice form),

- baking minimum at 180°C for 30 minutes (the first baking step),

- waiting for 1-2 hours for cooling down the cake,

- slicing the cake after cooling down,

- turning on the cooled oven again at 120°C, but with the fan turned off and the upper and lower heat turned on,

- baking the cake slices in the oven at 120°C for 3 more hours (the second baking step)

- turning upside down slices every half hour,

- after 3 hours, taking out the cake and leaving to cool down,

- pounding the slices in a mortar and ground into a powder like breadcrumbs.

8. A hypoallergenic nutritional milk product obtained by the method of any of the preceding claims.

9. The hypoallergenic nutritional milk product according to claim 9, wherein the said milk product is selected from the group consisting of a cake, a cookie, a muffin and a brownie.